The characteristics of the cytolytic toxin streptolysin O (SLO) have been studied for many years, but the nature of its lytic mechanism remains unknown. The purpose of this investigation is to study the action of SLO on erythrocyte membranes, emphasizing the effect of the toxin on membrane proteins. Membranes from normal and SLO-treated erythrocytes will be solubilized with sodium dodecyl sulfate and the polypeptide profiles examined with polyacrylamide gel electrophoresis; alterations in major or minor membrane polypeptides produced by SLO should be readily apparent. The effect of streptolysin O on the largest membrane proteins (molecular weight 240,000-320,000) will be tested directly by extracting these polypeptides in low ionic strength solutions, and treating them with toxin. The effect on membrane proteins of low concentrations of SLO at 37 degrees C and very high concentrations at O degrees C will be studied to see whether SLO has the same mechanism of action at the two temperatures. The role of cellular nicotinamide adenine dinucleotide (NAD) in the hemolytic process will be examined by determining whether the addition of NAD prevents or reverses the SLO-induced alterations in the membrane. Finally, the possibility that SLO is a proteolytic enzyme, activated by erythrocyte membrane proteases, will be investigated.